Unit

UNIT 10.8 Immunoblotting and Immunodetection

  1. Sean Gallagher1,
  2. Scott E. Winston (tank transfer systems)2,
  3. Steven A. Fuller (tank transfer systems)2,
  4. John G.R. Hurrell (tank transfer systems; reversible staining of proteins)3

Published Online: 1 MAY 2004

DOI: 10.1002/0471142727.mb1008s66

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Gallagher, S., Winston, S. E., Fuller, S. A. and Hurrell, J. G. 2004. Immunoblotting and Immunodetection. Current Protocols in Molecular Biology. 66:III:10.8:10.8.1–10.8.24.

Author Information

  1. 1

    UVP, Inc., Upland, California

  2. 2

    Nabi, Rockville, Maryland

  3. 3

    FluorRx, Carmel, Indiana

Publication History

  1. Published Online: 1 MAY 2004
  2. Published Print: APR 2004

This is not the most recent version of the article. View current version (1 JUL 2008)

Abstract

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

Keywords:

  • immunoblot;
  • western blot;
  • horseradish peroxidase;
  • alkaline phosphatase;
  • antibodies